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Advanced nanoscale antimicrobials, originated from the combination of noble metal nanoparticles (NPs) with conventional antimi-crobial drugs, are considered the next generation of antimicrobial agents. Therefore, there is an increasing demand for rapid, eco-friendly, and relatively inexpensive synthetic approaches for the preparation of nontoxic metallic nanostructures endowed with unique physicochemical properties. Recently, we have proposed a straightforward synthetic strategy that exploits the properties of polymeric beta-cyclodextrin (PolyCD) to act as both the reducing and stabilizing agent to produce monodispersed and stable gold-based NPs either as monometallic (nanoG) structures or core-shell bimetallic (nanoGS) architectures with an external silver layer. Here, we describe the preparation of a supramolecular assembly between nanoGS and pentamidine, an antileishmanial drug endowed with a wide range of therapeutic properties (i.e., antimicrobial, anti-inflammatory, and anticancer). The physicochemical characterization of the supramolecular assembly (nanoGSP) in terms of size and colloidal stability was investigated by complemen-tary spectroscopic techniques, such as UV-vis, zeta-potential, and dynamic light scattering (DLS). Furthermore, the role of PolyCD during the reduction/stabilization of metal NPs was investigated for the first time by NMR spectroscopy.

Type II NADH dehydrogenase (NDH-2) is an essential component of electron transfer in many microbial pathogens but has remained largely unexplored as a potential drug target. Previously, quinolinyl pyrimidines were shown to inhibit Mycobacterium tuberculosis NDH-2, as well as the growth of the bacteria [Shirude, P. S.; ACS Med. Chem. Lett. 2012, 3, 736−740]. Here, we synthesized a number of novel quinolinyl pyrimidines and investigated their properties. In terms of inhibition of the NDH-2 enzymes from M. tuberculosis and Mycobacterium smegmatis, the best compounds were of similar potency to previously reported inhibitors of the same class (half-maximal inhibitory concentration (IC50) values in the low-μM range). However, a number of the compounds had much better activity against Gram-negative pathogens, with minimum inhibitory concentrations (MICs) as low as 2 μg/mL. Multivariate analyses (partial least-squares (PLS) and principle component analysis (PCA)) showed that overall ligand charge was one of the most important factors in determining antibacterial activity, with patterns that varied depending on the particular bacterial species. In some cases (e.g., mycobacteria), there was a clear correlation between the IC50 values and the observed MICs, while in other instances, no such correlation was evident. When tested against a panel of protozoan parasites, the compounds failed to show activity that was not linked to cytotoxicity. Further, a strong correlation between hydrophobicity (estimated as clog P) and cytotoxicity was revealed; more hydrophobic analogues were more cytotoxic. By contrast, antibacterial MIC values and cytotoxicity were not well correlated, suggesting that the quinolinyl pyrimidines can be optimized further as antimicrobial agents. Lu Lu, Linda Åkerbladh and Shabbir Ahmad contributed equally to this work

The phosphodiesterase inhibitor tetrahydrophthalazinone NPD-008 was explored by phenotypic in vitro screening, target validation, and ultrastructural approaches against Trypanosoma cruzi . NPD-008 displayed activity against different forms and strains of T. cruzi (50% effective concentration [EC 50 ], 6.6 to 39.5 μM). Increased cAMP levels of T. cruzi and its combination with benznidazole gave synergistic interaction.

Approximately 20% of sleeping sickness patients exhibit respiratory complications, however, with a largely unknown role of the parasite. Here we show that tsetse fly-transmitted Trypanosoma brucei parasites rapidly and permanently colonize the lungs and occupy the extravascular spaces surrounding the blood vessels of the alveoli and bronchi. They are present as nests of multiplying parasites exhibiting close interactions with collagen and active secretion of extracellular vesicles. The local immune response shows a substantial increase of monocytes, macrophages, dendritic cells and gamma delta and activated alpha beta T cells and a later influx of neutrophils. Interestingly, parasite presence results in a significant reduction of B cells, eosinophils and natural killer cells. T. brucei infected mice show no infection-associated pulmonary dysfunction, mirroring the limited pulmonary clinical complications during sleeping sickness. However, the substantial reduction of the various immune cells may render individuals more susceptible to opportunistic infections, as evident by a co-infection experiment with respiratory syncytial virus. Collectively, these observations provide insights into a largely overlooked target organ, and may trigger new diagnostic and supportive therapeutic approaches for sleeping sickness. A number of human African trypanosomiasis, or sleeping sickness, patients suffer from respiratory symptoms commonly attributed to cardiac insufficiency. Here, the authors characterise the role of pulmonary Trypanosoma brucei in respiratory infection.

Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation. ispartof: Scientific Reports vol:10 issue:1 ispartof: location:England status: published

Objectives This study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate. Methods Experimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159LiROS3) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well. Results The fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3-reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse. Conclusion The natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain., Graphical abstract Image 1, Highlights • A clinical MIL-R L. infantum strain displays a loss-of-fitness in the mammalian host. • ROS3-deficiency is compatible with efficient transmission by two sand fly species. • Resistance is stable after mouse and sand fly passage. • Transmission of this clinical MIL-R strain is a risk for immunocompromised patients.

© The Author(s) 2020., [Background]: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure., [Methods]: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis., [Results]: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector., [Conclusions]: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector., Funding was available through the Research Fund Flanders (G051812N, 12I0317N and 1189617N) and a research fund of the University of Antwerp (TT-ZAPBOF 33049 and TOP-BOF 35017). LR was supported by Redes de Investigación Cooperativa RETICS-FEDER RD16/0027/0010.

13 p.-7 fig.-5 tab.-1 graph. abst., A previous phenotypic screening campaign led to the identification of a quinazoline derivative with promising in vitro activity against Schistosoma mansoni. Follow-up studies of the antischistosomal potential of this candidate are presented here. The in vivo studies in a S. mansoni mouse model show a significant reduction of total worms and a complete disappearance of immature eggs when administered concomitantly with praziquantel in comparison with the administration of praziquantel alone. This fact is of utmost importance because eggs are responsible for the pathology and transmission of the disease. Subsequently, the chemical optimisation of the structure in order to improve the metabolic stability of the parent compound was carried out leading to derivatives with improved drug-like properties. Additionally, the putative target of this new class of antischistosomal compounds was envisaged by using computational tools and the binding mode to the target enzyme, aldose reductase, was proposed., This study received funding from the EC 7th Framework Programme (FP7-HEALTH-2013-INNOVATION-1, PDE4NPD no.602666), RICET (RD16/0027/0010), FEDER funds and MECD [Grant FPU15/1465 to V. S.-P.].

Animal trypanosomiasis (AT) is a parasitic disease with high socio-economic impact. Given the limited therapeutic options and problems of toxicity and drug resistance, this study assessed redirecting our previously identified antitrypanosomal nucleosides for the treatment of AT. Promising hits were identified with excellent in vitro activity across all important animal trypanosome species. Compound 7, an inosine analogue, and our previously described lead compound, 3???-deoxytubercidin (8), showed broad spectrum anti-AT activity, metabolic stability in the target host species and absence of toxicity, but with variable efficacy ranging from limited activity to full cure in mouse models of Trypanosoma congolense and T. vivax infection. Several compounds show promise against T. evansi (surra) and T. equiperdum (dourine). Given the preferred target product profile for a broadspectrum compound against AT, this study emphasizes the need to include T. vivax in the screening cascade given its divergent susceptibility profile and provides a basis for lead optimization towards such broad spectrum anti-AT compound.

Long-term hematopoietic stem cells may act as protective niches for the Leishmania parasite, potentially contributing to treatment failure in cases of visceral leishmaniasis. Given the discontinuation of various first-line drugs for visceral leishmaniasis (VL), large-scale in vivo drug screening, establishment of a relapse model in rodents, immunophenotyping, and transcriptomics were combined to study persistent infections and therapeutic failure. Double bioluminescent/fluorescent Leishmania infantum and L. donovani reporter lines enabled the identification of long-term hematopoietic stem cells (LT-HSC) as a niche in the bone marrow with remarkably high parasite burdens, a feature confirmed for human hematopoietic stem cells (hHSPC). LT-HSC are more tolerant to antileishmanial drug action and serve as source of relapse. A unique transcriptional 'StemLeish' signature in these cells was defined by upregulated TNF/NF-kappa B and RGS1/TGF-beta/SMAD/SKIL signaling, and a downregulated oxidative burst. Cross-species analyses demonstrated significant overlap with human VL and HIV co-infected blood transcriptomes. In summary, the identification of LT-HSC as a drug- and oxidative stress-resistant niche, undergoing a conserved transcriptional reprogramming underlying Leishmania persistence and treatment failure, may open therapeutic avenues for leishmaniasis.

Objectives To gain insight into the propagation of miltefosine (MIL) resistance in visceral leishmaniasis, this laboratory study explored development of resistant parasites with a defective miltefosine transporter (MT) in sand flies. Methods Infectivity, colonization of stomodeal valve and metacyclogenesis of a MIL-resistant (MIL-R) Leishmania infantum LEM3323 line with a defective MT were assessed in the natural sand fly vectors Phlebotomus perniciosus and Lutzomyia longipalpis. Given our recent description of partial drug dependency of the MT-deficient line, the impact of MIL pre-exposure on sand fly infectivity was explored as well. Results A significant reduction in sand fly infection, stomodeal valve colonization and differentiation into metacyclics (determined by a lower flagellum/cell body length ratio) was observed in both vectors for MIL-R as compared to the isogenic parent MIL-susceptible line. Re-introduction of the wildtype MT gene into MIL-R tended to partially rescue the capacity to infect sand flies. Pre-exposure to MIL did not alter infectivity of the MIL-R line. Conclusion The MIL resistant L. infantum LEM3323 line is significantly hampered in its development and transmissibility potential in two sand fly vector species. Additional studies are warranted to evaluate whether this applies to other visceral Leishmania parasites with acquired MIL-resistance., Graphical abstract Image 1

African trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3’-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3’-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3’-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness., Trypanosoma brucei relies on uptake and conversion of purines from the host, which constitutes a potential drug target. Here, Hulpia et al. combine structural elements from known trypanocidal nucleoside analogues and develop a potent trypanocide with curative activity in animal models of acute and late stage sleeping sickness.

Background The protozoan Trypanosoma cruzi is auxotrophic for purines and causes Chagas’ disease (CD), a neglected illness affecting >6 million people. Combining the 3-deoxyribofuranose part of cordycepin with the modified purine ring of a nucleoside ‘hit’ led to the discovery of 4-amino-5-(4-chlorophenyl)-N7-(3′-deoxy-β-d-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine (Cpd1), revealing promising anti-T. cruzi activity. Objectives To further evaluate Cpd1 in vitro and in vivo to fully assess its therapeutic potential against CD, covering cell culture sterilization through washout assays, drug combination with benznidazole and long-term administration in T. cruzi-infected mice. Results Although less susceptible to Cpd1 than amastigotes, trypomastigotes present an impaired capacity to successfully establish intracellular infection of cardiac cultures. Combination of benznidazole with Cpd1 indicated no interaction (additive effect) (FIC index = 0.72) while administration to mice at one-tenth of the optimal dose (2.5 mg/kg and 10 mg/kg for Cpd1 and benznidazole, respectively) suppressed parasitaemia but failed to avoid mortality. Long-term treatment (60 days) gave a rapid drop of the parasitaemia (>98% decline) and 100% mice survival but only 16% cure. In vitro washout experiments demonstrated that although parasite release into the supernatant of infected cardiac cultures was reduced by >94%, parasite recrudescence did occur after treatment. Conclusions Parasite recrudescence did occur after treatment corroborating the hypothesis of therapeutic failure due to subpopulations of dormant forms and/or genetic factors in persister parasites involved in natural drug resistance.

9 p.-5 fig.-2 tab., We report the evaluation of 265 compounds from a PDE-focused library for their antischistosomal activity, assessed in vitro using Schistosoma mansoni. Of the tested compounds, 171 (64%) displayed selective in vitro activity, with 16 causing worm hypermotility/spastic contractions and 41 inducing various degrees of worm killing at 100 μM, with the surviving worms displaying sluggish movement, worm unpairing and complete absence of eggs. The compounds that did not affect worm viability (n=72) induced a complete cessation of ovipositing. 82% of the compounds had an impact on male worms whereas female worms were barely affected. In vivo evaluation in S. mansoni-infected mice with the in vitro ‘hit’ NPD-0274 at 20 mg/kg/day orally for 5 days resulted in worm burden reductions of 29% and intestinal tissue egg load reduction of 35% at 10 days posttreatment.Combination of praziquantel (PZQ) at 10 mg/kg/day for 5 days with NPD-0274 or NPD-0298 resulted in significantly higher worm killing than PZQ alone, as well as a reduction in intestinal tissue egg load, disappearance of immature eggs and an increase in the number of dead eggs., This work is part of the PDE4NPD consortium supported by Framework Program 7 of the European Commission No: 602666.Funding from RICET/FEDER funds (RD16/0027/0010), and the Ministry of Education, Culture and Sport (MECD) of Spain (Grant FPU15/1465 to V. S.-P.) is also acknowledged. Y. Z. was supported by a grant of the Chinese Science Council; A. R. B. and I. J. P. dE. were supported by the Netherlands Science Foundation.

As over 6 million people are infected with Chagas disease and only limited therapeutic options are available, there is an urgent need for novel drugs. The involvement of cyclic nucleotide phosphodiesterases (PDE) in the lifecycle and biological fitness of a number of protozoan parasites has been described and several of these enzymes are thought to be viable drug targets. Within this context, a PDE-focused library was screened for its ability to affect the viability of Trypanosoma cruzi parasites. 5-(3-(Benzyloxy)-4-methoxyphenyl)-2-isopropyl-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (4), previously reported as a human PDE4 inhibitor, was identified as a hit. Upon optimization on three positions of the phenylpyrazolone scaffold, 2-isopropyl-5-(4-methoxy-3-(pyridin-3-yl)phenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (34) proved to be the most active compound against intracellular forms of T. cruzi (pIC 50 = 6.4) with a 100-fold selectivity with respect to toxicity toward human MRC-5 cells. Evaluation on different life stages and clinically relevant T. cruzi strains revealed that the phenylpyrazolones are not active against the bloodstream form of the Y strain but show submicromolar activity against the intracellular form of the Y- and Tulahuen strains as well as against the nitro-drug-resistant Colombiana strain. In vitro screening of phenylpyrazolones against TcrPDEB1, TcrPDEC, and TcrCYP51 showed that there was a poor correlation between enzyme inhibition and the observed phenotypic effect. Among the most potent compounds, both TcrCYP51 and non-TcrCYP51 inhibitors are identified, which were both equally able to inhibit T. cruzi in vitro.

Background Miltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency. Methodology/Principal findings To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production. Conclusions/Significance Differential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure., Author summary Visceral leishmaniasis is a neglected tropical disease that is fatal if left untreated. Miltefosine is currently the only oral drug available but is increasingly failing to cure patients, resulting in its discontinuation as first-line drug in some endemic areas. To understand these treatment failures, we investigated the complex interplay of the parasite with the host immune system in the presence and absence of miltefosine. Our data indicate that miltefosine-resistant Leishmania parasites become severely hampered in their in vivo infectivity, which could be attributed to the induction of a pronounced innate immune response. Interestingly, the infection deficit was partially restored in the presence of miltefosine. Our results further indicate that miltefosine can exacerbate infections with resistant parasites by reducing innate immune recognition. This study provides new insights into the complex interplay between parasite, drug and host and discloses an immune-related mechanism of treatment failure.

Several members of the 3′,5′-cyclic nucleotide phosphodiesterase (PDE) family play an essential role in cellular processes, which has labeled them as interesting targets for various diseases. The parasitic protozoan Trypanosoma brucei, causative agent of human African trypanosomiasis, contains several cyclic AMP specific PDEs from which TbrPDEB1 is validated as a drug target. The recent discovery of selective TbrPDEB1 inhibitors has increased their potential for a novel treatment for this disease. Compounds characterized by a rigid biphenyl tetrahydrophthalazinone core structure were used as starting point for the exploration of novel TbrPDEB1 inhibitors. Using a virtual screening campaign and structure-guided design, diaryl ether substituted phthalazinones were identified as novel TbrPDEB1 inhibitors with IC50 values around 1 μM against T. brucei. This study provides important structure-activity relationship (SAR) information for the future design of effective parasite-specific PDE inhibitors.

[Image: see text] Antitubercular 7-substituted 2-nitroimidazo[2,1-b][1,3]oxazines were previously shown to exhibit potent antileishmanial and antitrypanosomal activities, culminating in a new clinical investigational drug for visceral leishmaniasis (DNDI-0690). To offset development risks, we continued to seek further leads with divergent candidate profiles, especially analogues possessing greater aqueous solubility. Starting from an efficacious monoaryl derivative, replacement of the side chain ether linkage by novel amine, amide, and urea functionality was first explored; the former substitution was well-tolerated in vitro and in vivo but elicited marginal alterations to solubility (except through a less stable benzylamine), whereas the latter groups resulted in significant solubility improvements (up to 53-fold) but an antileishmanial potency reduction of at least 10-fold. Ultimately, we discovered that O-carbamate 66 offered a more optimal balance of increased solubility, suitable metabolic stability, excellent oral bioavailability (100%), and strong in vivo efficacy in a visceral leishmaniasis mouse model (97% parasite load reduction at 25 mg/kg).

Ethnopharmacological relevance Terminalia albida (Combretaceae), widely used in Guinean traditional medicine, showed promising activity against Plasmodium falciparum and Candida albicans in previous studies. Bioassay-guided fractionation was carried out in order to isolate the compounds responsible for these activities. Materials and methods Fractionation and isolation were performed by flash chromatography, followed by semi-preparative HPLC-DAD-MS. The structural elucidation of the isolated compounds was carried out by 1D and 2D NMR as well as HR-ESI-MS. Isolated compounds were evaluated against Plasmodium falciparum, Candida albicans, Staphylococcus aureus and Escherichia coli, and their cytotoxicity against MRC-5 cells was determined. Results Bioassay-guided fractionation of Terminalia albida root resulted in the isolation of 14 compounds (1–14), and their antimicrobial properties were evaluated. Pantolactone (1) (IC50 0.60 ± 0.03 μM) demonstrated significant activity against P. falciparum. Other compounds, including 3,4,3′-tri-O-methyl-ellagic acid (3), the triterpenes arjunolic acid (5), arjungenin (6), arjunic acid (7) and arjunglucoside II (10), and the phenol glycoside calophymembranside-B (14), were less active and showed IC50 values in the range 5–15 μM. None of the tested compound showed antibacterial or antifungal activity. Conclusion These results may explain at least in part the activity of the root extract of T. albida against P. falciparum.

Deployment of Wolbachia to mitigate dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) transmission is ongoing in 12 countries. One way to assess the efficacy of Wolbachia releases is to determine invasion rates within the wild population of Aedes aegypti following their release. Herein we evaluated the accuracy, sensitivity and specificity of the Near Infrared Spectroscopy (NIRS) in estimating the time post death, ZIKV-, CHIKV-, and Wolbachia-infection in trapped dead female Ae. aegypti mosquitoes over a period of 7 days. Regardless of the infection type, time post-death of mosquitoes was accurately predicted into four categories (fresh, 1 day old, 2–4 days old and 5–7 days old). Overall accuracies of 93.2, 97 and 90.3% were observed when NIRS was used to detect ZIKV, CHIKV and Wolbachia in dead Ae. aegypti female mosquitoes indicating NIRS could be potentially applied as a rapid and cost-effective arbovirus surveillance tool. However, field data is required to demonstrate the full capacity of NIRS for detecting these infections under field conditions., Santos et al. demonstrate that the Near Infrared Spectroscopy (NIRS) can accurately estimate the death time of trapped female Aedes aegypti and vector infection with Zika virus, Chikungunya virus, or Wolbachia in a 7-day trapping period. This study suggests that NIRS may provide an accurate and inexpensive tool that improves arbovirus surveillance systems.

Chagas disease and leishmaniasis are two poverty-related neglected tropical diseases that cause high mortality and morbidity. Current treatments suffer from severe limitations and novel, safer and more effective drugs are urgently needed. Both Trypanosoma cruzi and Leishmania are auxotrophic for purines and absolutely depend on uptake and assimilation of host purines. This led us to successfully explore purine nucleoside analogues as chemotherapeutic agents against these and other kinetoplastid infections. This study extensively explored the modification of the 6-amino group of tubercidin, a natural product with trypanocidal activity but unacceptable toxicity for clinical use. We found that mono-substitution of the amine with short alkyls elicits potent and selective antitrypanosomal and antileishmanial activity. The methyl analogue 15 displayed the best in vitro activity against both T. cruzi and L. infantum and high selectivity versus host cells. Oral administration for five consecutive days in an acute Chagas disease mouse model resulted in significantly reduced peak parasitemia levels (75, 89 and 96% with 12.5, 25 and 50 mg/kg/day, respectively). as well as increased animal survival rates with the lower doses (83 and 67% for 12.5 and 25 mg/kg/day, respectively).

Pyrazolones are heterocyclic compounds with interesting biological properties. Some derivatives inhibit phosphodiesterases (PDEs) and thereby increase the cellular concentration of cyclic AMP (cAMP), which plays a vital role in the control of metabolism in eukaryotic cells, including the protozoan Trypanosoma cruzi , the etiological agent of Chagas disease (CD), a major neglected tropical disease. In vitro phenotypic screening identified a 4-bromophenyl-dihydropyrazole dimer as an anti- T. cruzi hit and 17 novel pyrazolone analogues with variations on the phenyl ring were investigated in a panel of phenotypic laboratory models. Potent activity against the intracellular forms (Tulahuen and Y strains) was obtained with 50% effective concentration (EC 50 ) values within the 0.17 to 3.3 μM range. Although most were not active against bloodstream trypomastigotes, an altered morphology and loss of infectivity were observed. Pretreatment of the mammalian host cells with pyrazolones did not interfere with infection and proliferation, showing that the drug activity was not the result of changes to host cell metabolism. The pyrazolone NPD-227 increased the intracellular cAMP levels and was able to sterilize T. cruzi -infected cell cultures. Thus, due to its high potency and selectivity in vitro , and its additive interaction with benznidazole (Bz), NPD-227 was next assessed in the acute mouse model. Oral dosing for 5 days of NPD-227 at 10 mg/kg + Bz at 10 mg/kg not only reduced parasitemia (>87%) but also protected against mortality (>83% survival), hence demonstrating superiority to the monotherapy schemes. These data support these pyrazolone molecules as potential novel therapeutic alternatives for Chagas disease.

Previous investigation of the potent antileishmanial properties of antitubercular 7-substituted 2-nitroimidazo[2,1-b][1,3]oxazines with biaryl side chains led to our development of a new clinical candidate for visceral leishmaniasis (DNDI-0690). Within a collaborative backup program, a racemic monoaryl lead (3) possessing comparable activity in mice but a greater hERG liability formed the starting point for our pursuit of efficacious second generation analogues having good solubility and safety. Asymmetric synthesis and appraisal of its enantiomers first established that chiral preferences for in vivo efficacy were species dependent and that neither form afforded a reduced hERG risk. However, in line with our findings in a structurally related series, less lipophilic heteroaryl ethers provided significant solubility enhancements (up to 16-fold) and concomitantly attenuated hERG inhibition. One promising pyridine derivative (49) displayed 100% oral bioavailability in mice and delivered a 96% parasite burden reduction when dosed at 50 mg/kg in a Leishmania donovani mouse model of visceral leishmaniasis.

Type I interferons (IFNs) induced by an endogenousLeishmaniaRNA virus or exogenous viral infections have been shown to exacerbate infections with New World CutaneousLeishmaniaparasites, however, the impact of type I IFNs in visceralLeishmaniainfections and implicated mechanisms remain to be unraveled. This study assessed the impact of type I IFN on macrophage infection withL. infantumandL. donovaniand the implication of sialoadhesin (Siglec-1/CD169, Sn) as an IFN-inducible surface receptor. Stimulation of bone marrow-derived macrophages with type I IFN (IFN-alpha) significantly enhanced susceptibility to infection of reference laboratory strains and a set of recent clinical isolates. IFN-alpha particularly enhanced promastigote uptake. Enhanced macrophage susceptibility was linked to upregulated Sn surface expression as a major contributing factor to the infection exacerbating effect of IFN-alpha. Stimulation experiments in Sn-deficient macrophages, macrophage pretreatment with a monoclonal anti-Sn antibody or a novel bivalent anti-Sn nanobody and blocking of parasites with soluble Sn restored normal susceptibility levels. Infection of Sn-deficient mice with bioluminescentL. infantumpromastigotes revealed a moderate, strain-dependent role for Sn during visceral infection under the used experimental conditions. These data indicate that IFN-responsive Sn expression can enhance the susceptibility of macrophages to infection with visceralLeishmaniapromastigotes and that targeting of Sn may have some protective effects in early infection.

BackgroundIn eco-epidemiological studies,Leishmaniadetection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-LeishmaniaSYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection ofLeishmaniain field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts.MethodsField-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target forLeishmaniadetection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve.ResultsThe qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10−3and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.ConclusionsThis study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification ofLeishmaniain sand flies. The assay is inexpensive, sensitive and pan-Leishmaniaspecific, and accordingly an excellent assay for high-throughput screening in entomological research.

Ethnopharmacological relevance In Guinea, medicinal plants play an important role in the management of infectious diseases including urinary disorders, skin diseases and oral diseases. This study was carried out to collect medicinal plant species employed for the treatment of these diseases and to investigate their antimicrobial potential. Materials and methods Based on an ethnobotanical investigation carried out in three Guinean regions, 74 traditional healers and 28 herbalists were interviewed and medicinal plants were collected. The most quoted plant species were evaluated for their antimicrobial activities against Staphylococcus aureus, Escherichia coli, Candida albicans, and in addition against Plasmodium falciparum. Results A total of 112 plant species belonging to 102 genera distributed over 42 botanical families were inventoried. Among the selected plant species, promising activities against C. albicans were obtained for the methanolic extracts of the stem bark of Terminalia albida (IC50 1.2 μg/ml), the leaves of Tetracera alnifolia (IC50 1.6 μg/ml) and the root bark of Swartzia madagascariensis (IC50 7.8 μg/ml). The highest activity against S. aureus was obtained for the dichloromethane extracts of the leaves of Pavetta crassipes (IC50 8.5 μg/ml) and the root of Swartzia madagascariensis (IC50 12.8 μg/ml). Twenty one extracts, obtained from twelve plant species, were strongly active against Plasmodium falciparum, including the dichloromethane extracts of the root and stem bark of Terminalia albida root (IC50 0.6 and 0.8 μg/ml), the leaves of Landolphia heudelotii (IC50 0.5 μg/ml), the stem bark of Combretum paniculatum (IC50 0.4 μg/ml) and the leaves of Gardenia ternifolia (IC50 1.3 μg/ml). Conclusion The present study provides a comprehensive overview of medicinal plants employed by Guinean traditional healers for the treatment of various microbial diseases, including urinary disorders, skin diseases and oral diseases. Some of the studied plant species showed promising antimicrobial activity and could be considered as a potential source for the development of new antifungal and/or antimalarial agents.

Background Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed. Methodology The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection. Principal findings Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed. Conclusions/Significance The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies.

For the development of new and potent antimalarial drugs, we designed the virtual library with three points of randomization of novel [1,2,4]triazolo[4,3-a]pyridines bearing a sulfonamide fragment. The library of 1561 compounds has been investigated by both virtual screening and molecular docking methods using falcipain-2 as a target enzyme. 25 chosen hits were synthesized and evaluated for their antimalarial activity in vitro against Plasmodium falciparum. 3-Ethyl-N-(3-fluorobenzyl)-N-(4-methoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine-6-sulfonamide and 2-(3-chlorobenzyl)-8-(piperidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one showed in vitro good antimalarial activity with inhibitory concentration IC50 = 2.24 and 4.98 &mu, M, respectively. This new series of compounds may serve as a starting point for future antimalarial drug discovery programs.

The present study aimed to develop, characterize and evaluate the amphotericin B–loaded nanostructured lipid carriers (AmB-NLCs) for topical treatment of cutaneous leishmaniasis (CL) and vulvovaginal candidiasis (VVC). AmB-NLCs were characterized for particle size, zeta potential, encapsulation efficiency and surface morphology. Prepared NLCs were also characterized for in vitro drug release, ex vivo skin permeation and deposition before evaluating their in vitro and in vivo efficacy. Cytotoxicity of NLCs was assessed on MRC-5 cells, whereas skin irritation potential was evaluated in vivo using rats. Significant accumulation of drug in to the skin supported the topical application potential of drug-loaded NLCs. Encapsulation of AmB in NLCs resulted in enhanced in vitro potency against promastigotes and intracellular amastigotes of L. major JISH 118 (IC50 ± SEM = 0.02 ± 0.1 μM for both) compared with free drug (IC50 ± SEM = 0.15 ± 0.2 & 0.14 ± 0.0, respectively). Similar improved potency of AmB-NLCs was also observed for other Leishmania and fungal strains compared with drug solution. Topical application of AmB-NLCs on L. major–infected BALB/c mice caused a significant reduction in parasite burden per mg of lesion (65 × 108 ± 13) compared with the control group (> 167.8 × 108 ± 11). Topical AmB-NLCs gel demonstrated superior efficacy in the vaginal C. albicans rat model for VVC as compared with plain AmB gel. Moreover, results of in vitro cytotoxicity assay and in vivo skin irritation test confirmed AmB-NLCs to be non-toxic and safe for topical use. In conclusion, NLCs may have promising potential as carrier for topical treatment of various conditions of skin and mucosa.

Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development., Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development. Resistance selection was performed by successive exposure of Leishmania infantum promastigotes (in vitro) and intracellular amastigotes (both in vitro and in golden Syrian hamsters). The stability of the resistant phenotypes was assessed after passage in mice and Lutzomyia longipalpis sandflies. Whole-genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs), and somy changes. The potential role of efflux pumps (the MDR and MRP efflux pumps) in the development of resistance was assessed by coincubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine, and probenecid). Repeated drug exposure of amastigotes did not result in the emergence of drug resistance either in vitro or in vivo. Selection at the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index, 5.8 to 24.5). This phenotype proved to be unstable after in vivo passage in mice and sandflies, suggesting that nonfixed alterations are responsible for the elevated resistance. In line with this, single nucleotide polymorphisms and indels identified by whole-genome sequencing could not be directly linked to the decreased drug susceptibility. Copy number variations were absent, whereas somy changes were detected, which may have accounted for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. The selection performed does not suggest the rapid development of resistance against aminopyrazoles in the field. Karyotype changes may confer elevated levels of resistance, but these do not seem to be stable in the vertebrate and invertebrate hosts. MDR/MRP efflux pumps are not likely to significantly impact the activity of the aminopyrazole leads.

Leishmania parasites have the capacity to rapidly adapt to changing environments in their digenetic life cycle which alternates between a vertebrate and an invertebrate host. Emergence of resistance following drug exposure can evoke phenotypic alterations that affect several aspects of parasite fitness in both hosts. Current studies of the impact of resistance are mostly limited to interactions with the mammalian host and characterization of in vitro parasite growth and differentiation. Development in the vector and transmission capacity have been largely ignored. This review reflects on the impact of drug resistance on its spreading potential with specific focus on the use of the sand fly infection model to evaluate parasite development in the vector and the ensuing transmission potential of drug-resistant phenotypes.

In vitro growth (inhibition) assays have a dual application, either supporting the discovery of novel drugs or as a monitoring tool of drug resistance in patient isolates. From an experimental design point of view, both are quite different with regard to the infecting Leishmania species and strain, the wide variety of permissive host cells (primary cells versus cell lines), drug exposure times, detection methods and endpoint criteria. Recognizing the need for enhanced assay standardization to decrease interlaboratory variation and improve proper interpretation of results, a detailed description is given of the basic fundamental procedures and requirements for routine in vitro growth of Leishmania spp. with specific focus on the intracellular amastigote susceptibility assay. Although the described experimental procedures focus on visceral Leishmania species, the same assay principles may apply for the cutaneous species as well.

Chagas disease is the leading cause of cardiac-related mortality in Latin American countries where it is endemic. Trypanosoma cruzi, the disease-causing pathogen, is unable to synthesize purines de novo, necessitating salvage of preformed host purines. Therefore, purine and purine-nucleoside analogues might constitute an attractive source for identifying antitrypanosomal hits. In this study, structural elements of two purine-nucleoside analogues (i.e., cordycepin and a recently discovered 7-substituted 7-deazaadenosine) led to the identification of novel nucleoside analogues with potent in vitro activity. The structureactivity relationships of substituents at C-7 were investigated, ultimately leading to the selection of compound 5, with a C-7 para-chlorophenyl group, for in vivo evaluation. This derivative showed complete suppression of T. cruzi Y-strain blood parasitemia when orally administered twice daily for 5 days at 25 mg/kg and was able to protect infected mice from parasite-induced mortality. However, sterile cure by immunosuppression could not be demonstrated.

Current treatment options for visceral leishmaniasis have several drawbacks, and clinicians are confronted with an increasing number of treatment failures. To overcome this, the Drugs for Neglected Diseases initiative (DNDi) has invested in the development of novel antileishmanial leads, including a very promising class of oxaboroles. The mode of action/resistance of this series to Leishmania is still unknown and may be important for its further development and implementation. Repeated in vivo drug exposure and an in vitro selection procedure on both extracellular promastigote and intracellular amastigote stages were both unable to select for resistance. The use of specific inhibitors for ABC-transporters could not demonstrate the putative involvement of efflux pumps. Selection experiments and inhibitor studies, therefore, suggest that resistance to oxaboroles may not emerge readily in the field. The selection of a genome-wide cosmid library coupled to next-generation sequencing (Cos-seq) was used to identify resistance determinants and putative targets. This resulted in the identification of a highly enriched cosmid, harboring genes of chromosome 2 that confer a subtly increased resistance to the oxaboroles tested. Moderately enriched cosmids encompassing a region of chromosome 34 contained the cleavage and polyadenylation specificity factor (cpsf) gene, encoding the molecular target of several related benzoxaboroles in other organisms.

Background Host proteases have been shown to play important roles in many viral activities such as entry, uncoating, viral protein production and disease induction. Therefore, these cellular proteases are putative targets for the development of antivirals that inhibit their activity. Host proteases have been described to play essential roles in Ebola, HCV, HIV and influenza, such that specific protease inhibitors are able to reduce infection. RSV utilizes a host protease in its replication cycle but its potential as antiviral target is unknown. Therefore, we evaluated the effect of protease inhibitors on RSV infection. Methods To measure the sensitivity of RSV infection to protease inhibitors, cells were infected with RSV and incubated for 18 h in the presence or absence of the inhibitors. Cells were fixed, stained and studied using fluorescence microscopy. Results Several protease inhibitors, representing different classes of proteases (AEBSF, Pepstatin A, E-64, TPCK, PMSF and aprotinin), were tested for inhibitory effects on an RSV A2 infection of HEp-2 cells. Different treatment durations, ranging from 1 h prior to inoculation and continuing for 18 h during the assay, were evaluated. Of all the inhibitors tested, AEBSF and TPCK significantly decreased RSV infection. To ascertain that the observed effect of AEBSF was not a specific feature related to HEp-2 cells, A549 and BEAS-2B cells were also used. Similar to HEp-2, an almost complete block in the number of RSV infected cells after 18 h of incubation was observed and the effect was dose-dependent. To gain insight into the mechanism of this inhibition, AEBSF treatment was applied during different phases of an infection cycle (pre-, peri- and post-inoculation treatment). The results from these experiments indicate that AEBSF is mainly active during the early entry phase of RSV. The inhibitory effect was also observed with other RSV isolates A1998/3–2 and A2000/3–4, suggesting that this is a general feature of RSV. Conclusion RSV infection can be inhibited by broad serine protease inhibitors, AEBSF and TPCK. We confirmed that AEBSF inhibition is independent of the cell line used or RSV strain. The time point at which treatment with the inhibitor was most potent, was found to coincide with the expected moment of entry of the virion with the host cell.

Respiratory Syncytial Virus (RSV) is a very important viral pathogen in children, immunocompromised and cardiopulmonary diseased patients and the elderly. Most of the published research with RSV was performed on RSV Long and RSV A2, isolated in 1956 and 1961, yet recent RSV isolates differ from these prototype strains. Additionally, these viruses have been serially passaged in cell culture, which may result in adaptations that affect virus&ndash, host interactions. We have isolated RSV from mucosal secretions of 12 patients in the winters 2016&ndash, 2017 and 2017&ndash, 2018, of which eight RSV-A subtypes and four RSV-B subtypes. Passage 3 of the isolates was assessed for viral replication kinetics and infectious virus production in HEp-2, A549 and BEAS-2B cells, thermal stability at 37 &deg, C, 32 &deg, C and 4 &deg, C, syncytia formation, neutralization by palivizumab and mucin mRNA expression in infected A549 cells. We observed that viruses isolated in one RSV season show differences on the tested assays. Furthermore, comparison with RSV A2 and RSV B1 reveals for some RSV isolates differences in viral replication kinetics, thermal stability and fusion capacity. Major differences are, however, not observed and differences between the recent isolates and reference strains is, overall, similar to the observed variation in between the recent isolates. One clinical isolate (BE/ANT-A11/17) replicated very efficiently in all cell lines, and remarkably, even better than RSV A2 in the HEp-2 cell line.

The structure-activity relationship of a 4-Azaindole-2-piperidine compound selected from GlaxoSmithKline’s recently disclosed open-resource “Chagas box” and possessing moderate activity against Trypanosoma cruzi, the parasite responsible for Chagas disease, is presented. Despite considerable medicinal chemistry efforts, a suitably potent and metabolically stable compound could not be identified to advance the series into in vivo studies. This research should be of interest to those in the area of neglected diseases and in particular anti-kinetoplastid drug discovery.

Background Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, needs urgent alternative therapeutic options as the treatments currently available display severe limitations, mainly related to efficacy and toxicity. Objectives As phosphodiesterases (PDEs) have been claimed as novel targets against T. cruzi, our aim was to evaluate the biological aspects of 12 new phthalazinone PDE inhibitors against different T. cruzi strains and parasite forms relevant for human infection. Methods In vitro trypanocidal activity of the inhibitors was assessed alone and in combination with benznidazole. Their effects on parasite ultrastructural and cAMP levels were determined. PDE mRNA levels from the different T. cruzi forms were measured by quantitative reverse transcription PCR. Results Five TcrPDEs were found to be expressed in all parasite stages. Four compounds displayed strong effects against intracellular amastigotes. Against bloodstream trypomastigotes (BTs), three were at least as potent as benznidazole. In vitro combination therapy with one of the most active inhibitors on both parasite forms (NPD-040) plus benznidazole demonstrated a quite synergistic profile (xΣ FICI = 0.58) against intracellular amastigotes but no interaction (xΣ FICI = 1.27) when BTs were assayed. BTs treated with NPD-040 presented disrupted Golgi apparatus, a swollen flagellar pocket and signs of autophagy. cAMP measurements of untreated parasites showed that amastigotes have higher ability to efflux this second messenger than BTs. NPD-001 and NPD-040 increase the intracellular cAMP content in both BTs and amastigotes, which is also released into the extracellular milieu. Conclusions The findings demonstrate the potential of PDE inhibitors as anti-T. cruzi drug candidates.

25 p.-4 fig.-2 tab., More than 100 years after being first described, Chagas disease remains endemic in 21 Latin American countries and has spread to other continents. Indeed, this disease, which is caused by the protozoan parasite Trypanosoma cruzi, is no longer just a problem for the American continents but has become a global health threat. Current therapies, i.e., nifurtimox and benznidazole (Bz), are far from being adequate, due to their undesirable effects and their lack of efficacy in the chronic phases of the disease. In this work, we present an in-depth phenotypic evaluation in T. cruzi of a new class of imidazole compounds, which were discovered in a previous phenotypic screen against different trypanosomatids and were designed as potential inhibitors of cAMP phosphodiesterases (PDEs). The confirmation of several activities similar or superior to that of Bz prompted a synthesis program of hit optimization and extended structure-activity relationship aimed at improving drug-like properties such as aqueous solubility, which resulted in additional hits with 50% inhibitory concentration (IC50) values similar to that of Bz. The cellular effects of one representative hit, compound 9, on bloodstream trypomastigotes were further investigated. Transmission electron microscopy revealed cellular changes, after just 2 h of incubation with the IC50 concentration, that were consistent with induced autophagy and osmotic stress, mechanisms previously linked to cAMP signaling. Compound 9 induced highly significant increases in both cellular and medium cAMP levels, confirming that inhibition of T. cruzi PDE(s) is part of its mechanism of action. The potent and selective activity of this imidazole-based PDE inhibitor class against T. cruzi constitutes a successful repurposing of research into inhibitors of mammalian PDEs., Funding from the EC 7th Framework Program (PDE4NPD, grant 602666), Red de Investigación Cooperativa en Enfermedades Tropicales (grant RD16/0027/0010), Fondo Europeo de Desarrollo Regional (FEDER), and Ministerio de Educación, Cultura y Deportes (grant FPU15/1465 to V.S.-P.) is acknowledged.

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide each year and there is presently no licensed vaccine to prevent severe RSV infections. Two major RSV glycoproteins, attachment (G) and fusion (F) protein, regulate viral replication and both proteins contain potential glycosylation sites which are highly variable for the G protein and conserved for the F protein among virus isolates. The RSV F sequence possesses five N-glycosylation sites located in the F2 subunit (N27 and N70), the p27 peptide (N116 and N126) and the F1 subunit (N500). The importance of RSV F N-glycosylation in virus replication and immunogenicity is not yet fully understood, and a better understanding may provide new insights for vaccine development. By using a BAC-based reverse genetics system, recombinant viruses expressing F proteins with loss of N-glycosylation sites were made. Mutant viruses with single N-glycosylation sites removed could be recovered, while this was not possible with the mutant with all N-glycosylation sites removed. Although the individual RSV F N-glycosylation sites were shown not to be essential for viral replication, they do contribute to the efficiency of in vitro and in vivo viral infection. To evaluate the role of N-glycosylation sites on RSV F antigenicity, serum antibody titers were determined after infection of BALB/c mice with RSV expressing the glycomutant F proteins. Infection with recombinant virus lacking the N-glycosylation site at position N116 (RSV F N116Q) resulted in significant higher neutralizing antibody titers compared to RSV F WT infection, which is surprising since this N-glycan is present in the p27 peptide which is assumed to be absent from the mature F protein in virions. Thus, single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches.

Leishmaniasis is a neglected parasitic disease for which the current antileishmania therapeutics are hampered by drug toxicity, high cost, need for parenteral administration, increasing treatment failure rates, and emergence of drug resistance. The R&D pipeline had run fairly dry for several years, but fortunately some new drug candidates are now under (pre)clinical development. Identification of novel drugs will nevertheless remain essential to adequately sustain and improve effective disease control in the future. In this review, a package of standard and accessible R&D approaches is discussed with expansion to some alternative strategies focusing on parasite-host and vector-host interactions.

(+)-N6-Hydroxyagelasine D, the enantiomer of the proposed structure of (−)-ageloxime D, as well as N6-hydroxyagelasine analogs were synthesized by selective N-7 alkylation of N6-[tert-butyl(dimethyl)silyloxy]-9-methyl-9H-purin-6-amine in order to install the terpenoid side chain, followed by fluoride mediated removal of the TBDMS-protecting group. N6-Hydroxyagelasine D and the analog carrying a geranylgeranyl side chain displayed profound antimicrobial activities against several pathogenic bacteria and protozoa and inhibited bacterial biofilm formation. However these compounds were also toxic towards mammalian fibroblast cells (MRC-5). The spectral data of N6-hydroxyagelasine D did not match those reported for ageloxime D before. Hence, a revised structure of ageloxime D was proposed. Basic hydrolysis of agelasine D gave (+)-N-[4-amino-6-(methylamino)pyrimidin-5-yl]-N-copalylformamide, a compound with spectral data in full agreement with those reported for (−)-ageloxime D.

Objectives: Miltefosine is currently the only oral drug for visceral leishmaniasis, and although deficiency in an aminophospholipidimiltefosine transporter (MT) is sufficient to elicit drug resistance, very few naturally miltefosine-resistant (MIL-R) strains have yet been isolated. This study aimed to make a detailed analysis of the impact of acquired miltefosine resistance and miltefosine treatment on in vivo infection. Methods: Bioluminescent versions of a MIL-R strain and its syngeneic parental line were generated by ntegration of the red-shifted firefly luciferase PpyRE9. The fitness of both lines was compared in vitro (growth rate, metacyclogenesis and macrophage infectivity) and in BALB/c mice through non-invasive bioluminescence imaging under conditions with and without drug pressure. Results: This study demonstrated a severe fitness loss of MT-deficient paras tes, resulting in a complete inability to multiply and cause a typical visceral leishmaniasis infection pattern in BALB/c mice. The observed fitness loss could not be rescued by host immune suppression with cyclophosphamide, whereas episomal reconstitution with a wild-type MT restored parasite virulence, hence linking parasite fitness to MT mutation. Remarkably, in vivo miltefosine treatment or in vitro miltefosine pre-exposure significantly rescued MIL-R parasite virulence. The in vitro pre-exposed MIL-R promastigotes showed a longer and more slender morphology, suggesting an altered membrane composition. Conclusions: The profound fitness loss of MT-deficient parasites most likely explains the low frequency of MIL-R clinical isolates. The observation that miltefosine can reverse this phenotype indicates a drug dependency of the MT-deficient parasites and emphasizes the importance of resistance profiling prior to miltefosine administration.

The nucleoside antibiotic tubercidin displays strong activity against different target organisms, but it is notoriously toxic to mammalian cells. However, the effects of tubercidin against T. brucei parasites inspired us to synthesize several C7 substituted analogs for in vitro evaluation, in order to find suitable hit compounds. C7 Deazaadenosines substituted with electron-poor phenyl groups were found to have micromolar activity against T. brucei in vitro. Replacement of the phenyl for a pyridine ring gave compound 13 with submicromolar potency and much-attenuated cytotoxicity compared to tubercidin. The veterinary pathogen T. congolense was equally affected by 13in vitro. Transporter studies in T. b. brucei indicated that 13 is taken up efficiently by both the P1 and P2 adenosine transporters, making the occurrence of transporter-related resistance and cross-resistance with diamidine drugs such as diminazene aceturate and pentamidine as well as with melaminophenyl arsenicals unlikely. Evaluation of the in vitro metabolic stability of analog 13 indicated that this analog was significantly metabolized in mouse microsomal fractions, precluding further in vivo evaluation in mouse models of HAT.

Ethnopharmacological relevance: An aqueous decoction of root bark of Alstonia congensis Engl. (Apocynaceae) is used in several African countries to treat various ailments including malaria. Materials and methods: Extracts of different polarity and isolated constituents were tested in vitro for their antiplasmodial activity against the chloroquine-resistant strain Plasmodium falciparum K1 and the chloroquine-sensitive strain P. falciparum NF54A19A, as well as for their cytotoxic effects againt MRC-5 cells (human lung fibroblasts). Extracts and fractions were evaluated in vivo against the chloroquine-resistant strain P. yoelii N67 and the chloroquine-sensitive strain P. berghei berghei ANKA. Results: The aqueous extract, the 80% methanol extract and the alkaloid-enriched extract exhibited strong antiplasmodial activity against P. falciparum K1 with IC50 values < 10 mu g/ml and against P. falciparum NF54 A19A with IC50 values < 0.02 mu g/ml. In vivo against P. yoelii N67, at the highest oral dose of 400 mg/kg body weight, all extracts produced 70-73% chemosuppression, while against P. berghei berghei, more than 75% chemosuppression was observed. With regard to the isolated constituents, boonein was inactive in vitro against P. falci-parwn K-1 (IC50 > 64 mu M), while echitamine, 6,7-seco-angustilobine B and beta-amyrin exhibited moderate activity (IC50 < 30 mu M). Against P. falciparum NF54 A19A, boonein was inactive (IC50 > 64 mu M), while echitamine, 6,7-secoangustilobine and beta-amyrin showed moderate IC50 values of 11.07, 21.26 and 40.70 mu M, respectively. Conclusion: These results demonstrated that all extracts from A. congensis root bark possessed antiplasmodial activity in vitro and in vivo. They can be used as raw materials for the preparation of ameliorated remedies for the treatment of uncomplicated malaria. The observed antiplasmodial activity may be due in part to the presence of indole alkaloids.

Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets. The activity of known nucleoside analogues such as tubercidin and cordycepin led to the development of a series of C7-substituted nucleoside analogues. Here, we use RNA interference (RNAi) libraries to gain insight into the mode-of-action of these novel nucleoside analogues. Whole-genome RNAi screening revealed the involvement of adenosine kinase and 4E interacting protein into the mode-of-action of certain antitrypanosomal nucleoside analogues. Using RNAi lines and gene-deficient parasites, 4E interacting protein was found to be essential for parasite growth and infectivity in the vertebrate host. The essential nature of this gene product and involvement in the activity of certain nucleoside analogues indicates that it represents a potential novel drug target.

The discovery of novel antiparasitic drugs for neglected tropical diseases (NTDs) constitutes a global urgency and requires a range of innovative strategies to ensure a sustainable pipeline of lead compounds. Thus far, primaquine (PQ) is the only transmission-blocking antimalarial that is clinically available, displaying marked activity against gametocytes of all causative species of human malaria (Plasmodium spp.). Chagas disease, caused by Trypanosoma cruzi, is another PQ-sensitive illness besides malaria. One of the major drawbacks of PQ is its metabolism into carboxyprimaquine (CPQ), which is less active than the parent drug. In this study, we developed different synthetic pathways to confer N-protection to PQ through introduction of thioxo-imidazolidin-4-one. The introduction of this group prevents the formation of CPQ, counteracting one major drawback of the parent drug. After that, we evaluated the potential biological activity of the novel 2-thioxo-imidazolidin-4-one derivative of PQ, which showed relevant in vitro activity against Trypanosoma cruzi (IC50 1.4 μM) compared to PQ (IC50 1.7 μM) and the reference drug benznidazole (IC50 1.6 μM). Noting its acceptable pharmacokinetic profile, this PQ conjugate may be a potential scaffold for novel drug exploration against Chagas disease.

The bioactive petroleum ether fraction of Verbesina encelioides, previously studied by the authors, was chosen for the isolation of antiprotozoal metabolites. Pseudotaraxasterol-3β-acetate (1), benzyl 2,6-dimethoxy benzoate (2), 16β-hydroxy-pseudotaraxasterol-3β-palmitate (3) and pseudotaraxasterol (4), in addition to β-sitosterol glucoside (5) and β-sitosterol galactoside (6) were isolated and identified based on one-dimensional and two-dimensional spectral analysis. This is the first report describing (3) and (6) in genus Verbesina. The isolated compounds were tested in vitro against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Cytotoxicity was evaluated on MRC-5 cells. Compound 1 showed moderate to weak activity against L. infantum T. brucei and P. falciparum and was inactive against T. cruzi. Compound 3 showed moderate activity against L. infantum, compound 4 revealed weak activity against T. cruzi, while 5 and 6 were inactive against all tested protozoa. All compounds were non-cytotoxic. The isolated constituents showed less antiprotozoal activity than the crude fraction.

Although miltefosine (MIL) has only been approved for the treatment of visceral leishmaniasis (VL) in 2002, its application in monotherapy already led to the development of two confirmed MIL-resistant isolates by 2009. Although liposomal amphotericin B is recommended as first-line treatment in Europe, MIL is still occasionally used in HIV co-infected patients. Since their immune system is incapable of controlling the infection, high parasite burdens and post-treatment relapses are common. Linked to the particular pharmacokinetic profile of MIL, successive treatment of recurrent relapses could in principle facilitate the emergence of drug resistance. This study evaluated the effect of immunosuppression (cyclophosphamide 150 mg/kg once weekly) on the development of MIL-resistance in Syrian golden hamsters infected with Leishmania infantum. The hamsters were treated with MIL (20 mg/kg orally for 5 days) whenever clinical signs of infection or relapse were observed. The immunosuppression resulted in a significant depletion of CD4+ lymphocytes and MHCII-expressing cells in peripheral blood, and a concomitant increase in tissue parasite burdens and shorter time to relapse, but the strain's susceptibility upon repeated MIL exposure remained unaltered. This study demonstrates that immunosuppression accelerates the occurrence of relapse without expediting MIL resistance development., Graphical abstract Image 1, Highlights • Successive treatment cycles of L. infantum in the Syrian golden hamster model do not result in MIL resistance. • Immunosuppression of infected hamsters with cyclophosphamide (CPA) does not expedite the emergence of MIL resistance. • Weekly CPA administration decreases the time-to-relapse and increases parasite burdens in liver, spleen and bone marrow. • Immunosuppression with CPA results in a depletion of CD4+ lymphocytes and MHCII-expressing cells in peripheral blood.

The available treatments for leishmaniasis are less than optimal due to inadequate efficacy, toxic side-effects and the emergence of resistant strains, clearly endorsing the urgent need for discovery and development of novel drug candidates. Ideally, these should act via an alternative mechanism-of-action to avoid cross-resistance with the current drugs. As cyclic nucleotide specific phosphodiesterases (PDEs) of L. major have been postulated as putative drug targets, a series of potential inhibitors of Leishmania PDEs was explored. Several displayed potent and selective in vitro activity against L. infantum intracellular amastigotes. One imidazole derivative, compound 35, was shown to reduce the parasite loads in vivo and dose-dependently increase the cellular cAMP level at just 2* and 5* the IC50, indicating a correlation between antileishmanial activity and increased cellular cAMP. Docking studies and molecular dynamics simulations pointed to imidazole 35 exerting its activity through PDE inhibition. This study establishes for the first time that inhibition of cAMP PDEs can potentially be exploited for new antileishmanial chemotherapy., Funding from the EC 7th Framework Programme (FP7-HEALTH-2013 INNOVATION-1, PDE4NPD no. 602666), MINECO (Grant SAF2015-65740-R), RICET (RD16/0027/0010), FEDER funds and MECD (Grant FPU15/1465 to V. S.-P.) is acknowledged.